【作者】 许海燕；

【导师】 焦新安；

【作者基本信息】 扬州大学 ， 遗传学， 2007， 硕士

【摘要】 弯曲菌(Campylobacter)是一种重要的食源性人兽共患病原菌,是全球范围内引起人胃肠道感染的最常见的病原菌之一,特定血清型空肠弯曲菌引起的肠炎是诱发格林-巴利综合症(GBS)的重要前驱感染因子。弯曲菌作为许多畜禽的寄生菌在自然界广泛存在,人常因食入被弯曲菌污染的食品等而感染。此外,由于弯曲菌耐药性的不断出现,食品安全和食源性疾病的问题已日益突出,成为公共卫生关注的新焦点。本研究的目的是:(1)对医院腹泻人群、牛群弯曲菌的感染流行状况进行初步调查研究;(2)应用国标方法鉴定分离株的生化特性,并应用2005年美国临床实验室标准化研究所(CLSI)推荐使用的琼脂扩散法进行耐药分析;(3)采用PCR-RFLP亚分型方法追溯不同来源的空肠弯曲菌间的相互关系。一、空肠弯曲菌和结肠弯曲菌的流行病学调查在2005年7月-2006年12月间,采集扬州市区三所医院门诊部和住院部的腹泻人群的粪便样品2,962份和江苏省10个奶牛场的产奶牛、育成牛、饲养环境样品1,531份,以国标方法为参照,采用增菌培养、选择性培养和多重PCR鉴定方法对上述样品进行空肠弯曲菌和结肠弯曲菌的分离鉴定。结果显示,在2,962份医院粪便样品中,143份空肠弯曲菌阳性,平均阳性率4.83%,10份结肠弯曲菌阳性,平均阳性率0.34%。统计分析表明,不同医院间感染率差异不显著(p>0.05),门诊部感染率极显著高于住院部(p<0.01),7岁以下幼儿感染率极显著高于其它年龄段人群(p<0.01),6月-8月感染率显著高于12月-2月和3月-5月份的感染率(p<0.05)。在1,531份牛场样品中,119份空肠弯曲菌阳性,平均阳性率7.77%;3份结肠弯曲菌阳性,平均阳性率0.20%;空肠弯曲菌、结肠弯曲菌阳性率最高分别为30.91%、3.57%。在三类样品中,产奶牛空肠弯曲菌、结肠弯曲菌的平均阳性率分别为5.02%、0.32%,育成牛空肠弯曲菌的平均阳性率为8.70%、未检出结肠弯曲菌,环境样品空肠弯曲菌、结肠弯曲菌的平均阳性率分别为10.28%、0.18%。上述结果显示,空肠弯曲菌和结肠弯曲菌在腹泻人群和奶牛群体的流行呈现多样化和复杂化,牛群的感染率高于人群,应加强牛群弯曲菌的控制,将有助于控制弯曲菌的感染和传播。二、人源和牛源弯曲菌分离株的生化特性与耐药性分析采用国标方法和2005年美国临床实验室标准化研究所(CLSI)推荐使用的琼脂扩散法对93株人源空肠弯曲菌、35株牛源空肠弯曲菌,以及13株结肠弯曲菌的生化特性和耐药性进行了研究。生化实验结果显示,128株空肠弯曲菌和13株结肠弯曲菌的氧化酶实验均呈阳性,128株空肠弯曲菌的马尿酸钠水解实验呈阳性,13株结肠弯曲菌的马尿酸钠水解实验呈阴性,结果表明所有的分离株均为弯曲菌,且本研究所分离的空肠弯曲菌在实验室培养条件下没有失去分解马尿酸盐的能力。药敏实验结果显示,93株人源空肠弯曲菌对8大类21种抗生素高度敏感的是:庆大霉素91.40%、阿齐红霉素89.25%、红霉素88.46%、阿莫西林87.10%、链霉素83.87%、头孢噻肟81.72%、卡那霉素81.72%,高度耐药的是:头孢哌酮100%、复方新诺明100%、头孢拉定97.85%、头孢克罗93.55%、环丙沙星91.40%、萘啶酸91.40%、左旋氧氟沙星88.17%、土霉素87.50%。耐药菌株的耐药谱主要集中在9耐至14耐,达81.71%,两医院分离株多重耐药性无显著性差异(p>0.05),不同诊区分离株的多重耐药性间存在显著性差异(p<0.05)。35株牛源空肠弯曲菌对抗生素高度敏感的是:阿莫西林100%、阿齐红霉100%、链霉素97.14%、庆大霉素94.29%、红霉素91.43%、头孢噻肟82.86%、克林霉素82.86%;高度耐药的是:头孢哌酮100%、恩诺沙星100%、环丙沙星97.14%、复方新诺明97.14%、左旋氧氟沙星94.29%、萘啶酸94.29%、头孢拉定94.29%、诺氟沙星91.43%、头孢克罗88.57%。菌株耐药谱显示,35株分离株的耐药主要集中在9耐到12耐,占88.57%,产奶牛分离株的多重耐药性较其它来源的分离株的耐药性更严重。13株结肠弯曲菌对临床常用的抗生素高度敏感的是:头孢噻肟100.00%、阿莫西林92.31%、红霉素92.31%、阿齐红霉素92.31%、克林霉素84.62%,高度耐药的是:头孢拉定100%、头孢哌酮100%、诺氟沙星100%、恩诺沙星100%、环丙沙星100%、左旋氧氟沙星100%、萘定酮酸100%、复方新诺明100%、四环素100%、头孢克罗92.31%、强力霉素92.31%、土霉素88.89%。耐药菌株的耐药谱主要分布在16耐、15耐、13耐和11耐,占76.91%。与空肠弯曲菌的耐药谱相比,结肠弯曲菌的多重耐药性较为严重。人源和牛源的分离株对头孢菌素类、喹诺酮类和磺胺类抗生素均产生了很强的耐药性,此外,人源分离株还对四环素类抗生素产生了较强的耐药性,而且多重耐药的现象很严重,92.2%的菌株在9耐以上,更值得人们注意的是,通过对临床用药的分析,弯曲菌的耐药性并非是完全在治疗弯曲菌病的过程中产生的,主要是在用抗生素防止和治疗其它各种感染的过程中产生的,弯曲菌的耐药性多属于被动耐药。因此在抗生素的使用过程中,要注意交叉用药,防止多重耐药菌株的产生。同时,还必须严格控制各种耐药微生物到处传播,以防多重耐药性病原菌的交叉传递。我国人源和牛源空肠弯曲菌、结肠弯曲菌的耐药状况呈现多样化和复杂化,必须加强各地弯曲菌耐药性和抗生素使用情况的监测,及时调整用药方案和公共卫生策略。三、空肠弯曲菌FlaA基因RFLP分子亚分型方法的建立与应用根据文献合成引物,PCR扩增目的片段,限制性内切酶DdeⅠ进行酶切,建立PCR-RFLP亚分型方法,重复性实验表明该方法具有良好的分型能力,是一种快速、简便、辨别力强、重复性好的分子亚分型方法,可作为研究空肠弯曲菌来源和传播途径的有力工具。应用该分型方法对329株不同来源的空肠弯曲菌进行分析,277株菌株得到了有效分型,可分型率为84.19%。通过对获得的图谱进行比对,将菌株分成了20个不同的RFLP谱型,优势谱型有2、5、6、7、9、11、12、15、16、18、20,占82.67%。其中,170株鸡群分离的空肠弯曲菌被分布在20个不同的谱型中,70株人源分离的菌株被分为19个不同的基因谱型,25株牛源空肠弯曲菌被分为13个不同的谱型,12株市场分离的菌株被分为6个基因型别。在人源的9个主要型别中,有8个分别与其它三种来源菌株的主要型别相互重叠。人源分离株的型别与鸡源、牛源以及市场禽肉分离株的型别呈交叉分布,表明牛、鸡是人类感染空肠弯曲菌的主要传染源之一,为研究空肠弯曲菌的种内差异和致病力差异奠定了基础。上述结果显示本文建立的空肠弯曲菌PCR-RFLP亚分型方法,对空肠弯曲菌感染源追踪有重要应用价值。更多还原

【Abstract】 Campylobacter is the most common zoonotic bacteria associated with human diarrhea in both developing and developed countries. Guillain-Barre syndrome is the most common acute flaccid paralysis due to an autoimmune disorder in nature. A considerable number of GBS patients present with a prior history of Campylobacteriosis, and GBS is considered a sequela of infections caused specifically by C. jejuni. Campylobacter is normally contracted through consumption of contaminated foods including those derived from food animals. Furthermore, an emerging public health concern regarding Campylobacter is the issue of microbial food safety due to increasing numbers of antimicrobial-resistant isolates. The objective of this study is to: (i) investigate the prevalence of C. jejuni and C. coli among diarrhea patients at Yangzhou and dairy cattle in Jiangsu Province; (ii) identify the isolates by phenotypic tests and detect the antimicrobial susceptibility with K-B method recommended by Clinical and Laboratory Standard Institute in 2005; (iii) track the source of C. jejuni isolates form different origins by PCR-RFLP.1. Prevalence of C. jejuni and C. coli among diarrhea patients and dairy cattleIn this study we attempted to investigate the prevalence of C. jejuni and C. coli among diarrhea patients at Yangzhou and dairy cattle in Jiangsu Province, 2,962 of human feces samples from three different hospitals at Yangzhou and 1,531 of cloaca samples from ten cattle farms were collected for the isolation and identification of C. jejuni and C. coli. The results showed that there were 143 strains of C. jejuni and 10 strains of C. coli isolated from 2,962 human feces samples, the average positive rate was 4.83% and 0.34% respectively. Outpatients and children≤7 years old had higher prevalence rate than that of inpatients (p<0.01). The detection rate of infection during June to August had higher prevalence rate than that of other months (p<0.05). 119 strains of C. jejuni and 3 strains of C. coli were isolated from 1,531 cloaca samples of 10 different cattle farms, the average positive rate was 7.77% and 0.20%, the highest positive rate of C. jejuni and C. coli was 30.91% and 3.57% respectively. The prevalence of C. jejuni in milk cow, heifer and environment samples was 5.02%, 8.70% and 10.28% respectively, and of C. coli was 0.32%, 0.00% and 0.18%, respectively. All results obtained from this study showed that endemic of Campylobacter spp. became diverse and complication in different colonies and provided well foundation for the right evaluation and established viable measures of Campylobacter spp..2. Antibiotic resistance of C. jejuni and C. coli isolated from humans and cattleThis study was undertaken to investigate the biological properties and antibiotic resistance of C. jejuni and C. coli from diarrheal samples and cloaca samples of cattle. All the isolates were positive in oxidation tests and the hippurate hydrolase of 128 C. jejuni were also positive, the hippurate hydrolase of all other 13 C.coli were negative. The results suggested that all the isolates were Campylobacters and all the isolates of C. jejuni didn’t lose the ability to hydrolyze hippurate.The 93 isolated C. jejuni from patients with sporadic diarrhea were analyzed for susceptibility to 21 kinds of antibiotics, and the results showed that these isolated strains were highly sensitive to seven kinds of antibiotics such as Gentamicin 91.40%, Azithromycin 89.25%, Erythromycin 88.46%, Amoxicillin 87.10%, Streptomycin 83.87%, Cefotaxime 81.72%, Kanamycin 81.72%, and were resistant to some frequently used medicines such as Cefoperazone 100%, Co-trimoxazole 100%, Cephalexin 97.85%, Cefaclor 93.55%, Ciprofloxacin 91.40%, Nalicixic acid 91.40%, Levofloxacin 88.17%, Oxytetracycline 87.50%. The percentage of isolates were 81.71% with resistance to 9 to 12 antimicrobials, and the multidrug resistance of isolated strains from outpatients was more severe than that of other isolated strains.Antibiotic resistance of 35 isolated C. jejuni strains from cattle were evaluated with 21 kinds of antibiotics, and the results showed that these isolated strains were highly sensitive to seven kinds of antibiotics such as Amoxicillin 100%, Azithromycin 100%, Streptomycin 97.14%, Gentamicin 94.29%, Erythromycin 91.43%, Cefotaxime 82.86%, Clindamycin 82.86%, and were resist to some frequently used medicines such as Cefoperazone 100%, Ofloxacin 100%, Ciprofloxacin 97.14%, Co-trimoxazole 97.14%, Levofloxacin 94.29%, Nalicixic acid 94.29%, Cephalexin 94.29%, Norfloxacin 91.43%, Cefaclor 88.57%. The percentage of isolates was 88.57% with resistance to 9 to 12 antimicrobials, and the multidrug resistance of isolated strains from cow was more severity than that of other isolated strains.Antibiotic resistance of 13 isolated C. coli strains were all evaluated by 21 kinds of antibiotics, and the results showed that these isolates were only sensitive to five kinds of antibiotics such as Cefotaxime 100%, Amoxicillin 92.31%, Erythromycin 92.31%, Azithromycin 92.31%, Clidamycin 84.62%, and were resistant to many frequently used antibiotics such as Cephalexin 100%, Cefoperazone 100%, Norfloxacin 100%, Ofloxacin 100%, Ciprofloxacin 100%, Levofloxacin 100%, Nalicixic acid 100%, Co-trimoxazole 100%, Tetracycline 100%, Cefaclor 92.31%, Doxycycline 92.31%, Oxytetracycline 88.89%. The percentage of isolates was 76.91% with resistance to 11, 13, 15, 16 kinds of antimicrobials, compared with the isolates of C. jejuni, the multidrug resistance of isolated C. coli was more severe.The results of this study showed that the isolated strains were resistant to a number of common antibiotics used in clinic. For instance, Cefoperazon, Cephalexin, Cefaclor, Ofloxacin, Co-trimoxazole, Ciprofloxacin, Nalicixic acid, Levofloxacin, Oxytetracycline and so on. The percentage of isolates was 92.2% with resistance to more than 9 kinds of antimicrobials. Worryingly, the multidrug resistant isolates were developed in the process of treating other kinds of infections, not Campylobacteriosis. So we should pay more attention to the development of antimicrobial resistance in Campylobacter spp. to prevent multidrug resistant isolates emerging. In a word, antimicrobial resistance of the Campylobacter spp. became more and more diverse and complicated in our country. Monitoring systems for Campylobacter spp. are important for inspecting the trends of antimicrobial susceptibility and supporting development of public policies for better prevention and treatment of Campylobacteriosis.3. Genotyping of C. jejuni isolates from different origins using PCR-fla-RFLP subtyping methodThe major objective of this work was to explore the genetic similarity between C. jejuni isolated from different sources. C. jejuni isolated from different sources (n=329) were subtyped by PCR-fla-RFLP. A flagellin gene fragment of 1.7kb was amplified from 277 C. jejuni isolates by PCR with a pair of fla gene specific primers. Digested with DdeⅠ, the PCR products showed twenty different banding profiles. Profile number 2, 5, 6, 7, 9, 11, 12, 15, 16, 18, 20 were the most frequently observed among different sources, accounting for 82.67%. In this paper, profile number 2 and 5 were predominant in the isolates from humans with sporadic diarrhea, retail poultry, bovine feces and poultry feces, which indicated that the genotype distribution of C. jejuni in poultry and bovine are similar to those of human Campylobacteriosis. We also observed that profile number 6, 11, 15, 18 were predominant in human and poultry isolates, while genotype 7 and 16 were predominant in human and bovine isolates. These findings indicated that genotype distribution in human isolates overlaps with genotype distribution in poultry and bovine isolates, presenting the possibility that bovine, as well as chickens, may serve as a source of human Campylobacteriosis. The results also show that PCR-fla-RFLP could be a powerful tool for the investigation of molecular epidemiological pattern of C. jejuni and for tracing the source of C. jejuni in clinical patients.更多还原